Microfluidic chromatography

ABSTRACT

The present invention is directed to a microfluidic chromatography apparatus comprising a microfabricated fluid delivery system and a chromatography column which is in fluid communication with the fluid delivery system, and a method for producing and using the same. Preferably, the chromatography column comprises an OTLC, PCLC, or combinations thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/281,937, filed Apr. 6, 2001.

FIELD OF THE INVENTION

The present invention relates to a microfluidic device comprising amicrofabricated fluid delivery system and a chromatography column. Inparticular, the present invention relates to a microfluidic devicecomprising an OTLC column, PCLC column, or combinations thereof, whichis operatively interconnected to a microfabricated fluid deliverysystem.

BACKGROUND OF THE INVENTION

Microfluidic devices allow manipulation of extremely small volumes ofliquids, and therefore are particularly useful in small scale samplepreparations, chemical synthesis, sample assay, sample screening, andother applications where a micro-scale amount of samples are involved.For many applications, the chemical make up of the resulting material(i.e., sample) needs to be analyzed. Such analysis typically requires atleast some degree of sample purification and/or separation. However, dueto the small sample size (e.g., nanoliter to microliter) used by thesemicrofluidic devices, conventional separation techniques are notapplicable.

Use of packed capillary and open tubular liquid chromatography (PCLC andOTLC, respectively) separation techniques have become increasinglypopular due to the demonstrated means of achieving high chromatographyefficiency with low operation pressures. Conventional high performanceliquid chromatography (i.e., HPLC) typically requires >2000 psipressure. In contrast, pressure of as low as 5 psi can be used for OTLCand PCLC. Some of the advantages of the OTLC and PCLC techniquesinclude, but are not limited to: (1) an increased efficiency, (2) alower sample dilution requirement, thereby increasing the sampledetection sensitivity, e.g., using a mass spectrometer, (3) a smalleramount of eluent requirement, and (4) the small sample amountrequirement. The latter advantage is of particularly importance in avariety of fields, such as proteomics, genomics, forensics, and otherareas where a minute quantity of sample is to be separated or purified.Unfortunately, in order to achieve the desired sensitivity andefficiency in OTLC and PCLC, the inner diameter of OTLC and PCLC columnsneed to be small, generally in the order of 50 μm or less, andpreferably about 10 μm or less. The small column diameter size in OTLCand PCLC techniques requires an equally precise sample injection andpumping system. To be effective, OTLC and PCLC techniques require asample flow rate of 0.01 μL/min or less. Conventional sample pumpingsystem can not adequately meet this stringent requirement. In addition,difficulties with large interconnection dead volume and detection volumebetween the OTLC or PCLC column and the fluid delivery (i.e., pumping)system have greatly limited the application of OTLC and PCLC techniques.

Therefore, there is a need for OTLC and PCLC devices which comprise asample injection and fluid pumping system that can achieve a sample flowrate of 0.01 μL/min or less. There is also a need for OTLC and PCLCdevices which have small or no dead volume between the OTLC or PCLCcolumn and the fluid delivery system.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a microfluidicchromatography apparatus for separating an analyte in a sample fluid.The microfluidic chromatography apparatus of the present inventioncomprises a microfabricated fluid delivery system and a chromatographycolumn. The microfabricated fluid delivery system of the presentinvention is capable of pumping a minute amount of fluid through thechromatography column. Preferably, the microfabricated fluid deliverysystem is capable of pumping (i.e., delivering or transporting) a fluidthrough the chromatography column at a flow rate of 0.01 μL/min or less.Thus, microfluidic chromatography apparatuses of the present inventionare particularly useful in separating analyte(s) from a minute quantityof sample fluid.

Preferably, the fluid delivery system of the present invention isproduced from a material comprising an elastomeric polymer. In oneparticular embodiment, the elastomeric polymer is selected from thegroup consisting of poly(carborane-siloxanes),poly(bis(fluoroalkoxy)phosphazene), poly(acrylonitrile-butadiene),poly(1-butene), poly(chlorotrifluoroethylene-vinylidene fluoride)copolymers, poly(ethyl vinyl ether), poly(vinylidene fluoride),poly(vinylidene fluoride-hexafluoropropylene) copolymer, elastomericpolyvinylchloride, polysulfone, polycarbonate, polymethylmethacrylate,polytertrafluoroethylene, polydimethylsiloxane, polydimethylsiloxanecopolymer, and aliphatic urethane diacrylate.

The fluid deliver system of the present invention comprises:

-   -   (i) a microfluidic flow channel comprising a flow channel inlet        for introducing the fluid into said flow channel and a flow        channel outlet,    -   (ii) a flow control channel,    -   (iii) a flow control valve comprised of a flow control        elastomeric segment that is disposed in between said flow        channel and said flow control channel to regulate fluid flow        through said flow channel, wherein said flow control valve is        deflectable into or retractable from said flow channel upon        which said flow control valve operates in response to an        actuation force applied to said flow control channel, said flow        control elastomeric segment when positioned in said flow channel        restricting fluid flow therethrough, and    -   (iv) a flow control channel actuation system operatively        interconnected to said flow control channel for applying an        actuation force to said flow control channel.

The fluid delivery system of the present invention can further compriseother component(s) depending on a particular need. For example, in oneparticular embodiment, the fluid delivery system further comprises aperistaltic pump which is comprised of one or more of the flow controlvalves.

The fluid delivery system can also comprise an eluent inlet which is influid communication with the flow channel inlet for introducing aneluent to said flow channel. In one specific embodiment, the eluentinlet further comprises:

-   -   an eluent reservoir comprising an eluent reservoir inlet        channel;    -   an eluent reservoir inlet control channel;    -   an eluent reservoir inlet control valve for opening and closing        fluid communication between said eluent reservoir and said flow        channel, wherein said eluent reservoir inlet control valve        comprises an elastomeric segment of said eluent reservoir inlet        control channel that is disposed in between said eluent        reservoir inlet control channel and said eluent reservoir inlet        channel to regulate fluid flow through said eluent reservoir        inlet channel, wherein said eluent reservoir inlet control valve        is deflectable into or retractable from said eluent reservoir        inlet channel upon which said eluent reservoir inlet control        valve operates in response to an actuation force applied to said        eluent reservoir inlet control channel, said elastomeric segment        of said eluent reservoir inlet control valve when positioned in        said eluent reservoir inlet channel restricting fluid flow        therethrough;    -   an eluent reservoir inlet control channel actuation system        operatively interconnected to said eluent reservoir inlet        control channel for applying an actuation force to said eluent        reservoir inlet control channel.

The flow channel inlet of the fluid delivery system can also comprise:

-   -   a sample reservoir comprising a sample reservoir inlet channel        which is in fluid communication with said flow channel;    -   a sample reservoir inlet control channel;    -   a sample reservoir inlet control valve for opening and closing        fluid communication between said sample reservoir and said flow        channel, wherein said sample reservoir inlet control valve        comprises an elastomeric segment of said sample reservoir inlet        control channel that is disposed in between said sample        reservoir control channel and said sample reservoir inlet        channel to regulate fluid flow through said sample reservoir        inlet channel, wherein said sample reservoir inlet control valve        is deflectable into or retractable from said sample reservoir        inlet channel upon which said sample reservoir inlet control        valve operates in response to an actuation force applied to said        sample reservoir inlet control channel, said elastomeric segment        of said sample reservoir inlet control channel when positioned        in said sample reservoir inlet channel restricting fluid flow        therethrough; and    -   an sample reservoir inlet control channel actuation system        operatively interconnected to said sample reservoir inlet        control channel for applying an actuation force to said sample        reservoir inlet control channel.

The chromatography column of the present invention comprises:

-   -   (i) a stationary phase which is capable of separating at least a        portion of the analyte from the sample fluid,    -   (ii) a column inlet which is in fluid communication with said        flow channel outlet, and    -   (iii) a column outlet through which a separated fluid exits the        chromatography column.

Preferably, the chromatography column is a separately fabricatedcomponent which is then integrated with the microfabricated fluiddelivery system. Advantages of this embodiment include the capability ofusing the microfabricated fluid delivery system with a variety ofdifferent chromatography columns and interchangeability ofchromatography columns depending on the need. Thus, in one particularembodiment, the chromatography column is a microfluidic chromatographydevice comprising a chromatography channel having an inner surface.Preferably, the stationary phase is covalently bonded to the innersurface of the chromatography channel. The stationary phase can bebonded to the chromatography column by a variety of means conventionallyknown to one skilled in the art. Such methods include activating ordepositing ions on the inner surface of the column. Preferably, thestationary phase is bonded to the inner surface of the column withoutthe need for any surface activation process. In this manner, anintegrated microfluidic chromatography system can be fabricated.

In one embodiment, the chromatography column comprises a microfabricatedrotary channel comprising:

-   -   a rotary channel inlet;    -   a rotary channel outlet;    -   a rotary control channel;    -   a rotary inlet control valve comprised of an elastomeric segment        of said rotary inlet control channel that is disposed in between        said rotary channel inlet and said rotary control channel to        regulate fluid flow into said rotary channel, wherein said        rotary inlet control valve is deflectable into or retractable        from said rotary channel inlet upon which said rotary inlet        control valve operates in response to an actuation force applied        to said rotary control channel, said elastomeric segment of said        rotary inlet control channel when positioned in said rotary        channel inlet restricting fluid flow therethrough;    -   a rotary outlet control valve comprised of an elastomeric        segment of said rotary outlet control channel that is disposed        in between said rotary channel outlet and said rotary control        channel to regulate fluid flow out of said rotary channel,        wherein said rotary outlet control valve is deflectable into or        retractable from said rotary channel outlet upon which said        rotary outlet control valve operates in response to an actuation        force applied to said rotary control channel, said elastomeric        segment of said rotary control channel outlet when positioned in        said rotary channel outlet restricting fluid flow therethrough;    -   a rotary pump valve comprised of an elastomeric segment of said        rotary pump that is disposed in between said rotary channel and        said rotary pump control channel to regulate fluid flow through        said rotary channel, wherein said rotary pump valve is        deflectable into or retractable from said rotary channel upon        which said rotary pump valve operates in response to an        actuation force applied to said rotary pump control channel,        said elastomeric segment of said rotary pump when positioned in        said rotary channel restricting fluid flow therethrough; and    -   a rotary control channel actuation system operatively        interconnected to said rotary control channel for applying an        actuation force to said rotary control channel.

In one particular embodiment, the chromatography column is an opentubular liquid chromatography column or a packed capillary liquid columnor a combination of these two columns.

The column outlet can also be in fluid communication with a sampledetection system inlet. In this manner, the fluid exiting thechromatography column can be analyzed directly with a detectionapparatus.

Furthermore, other components, such as sample preparation and detectioncomponents, can be fabricated or incorporated within the microfluidicchromatography apparatus of the present invention to provideparallel-processing systems.

In one embodiment of the present invention, the flow channel is locatedon an interface between a solid substrate and the elastomeric polymersuch that an inner surface of the flow channel comprises an elastomericpolymer portion and a solid substrate portion. In one particularembodiment, the stationary phase is attached to the solid substrateportion of the flow channel inner surface. In one specific embodiment,the elastomeric polymer portion of the flow channel inner surfacecomprises a surface coating that reduces a non-specific binding of theanalyte.

Another aspect of the present invention provides a method for producingthe microfluidic chromatography apparatus. In one particular embodiment,such a method comprises:

-   -   (a) producing a microfabricated fluid delivery system from a        material comprising an elastomeric polymer, wherein the fluid        deliver system comprises:        -   (i) a microfluidic flow channel comprising a flow channel            inlet for introducing the fluid into said flow channel and a            flow channel outlet,        -   (ii) a flow control channel,        -   (iii) a flow control valve comprised of a flow control            elastomeric segment that is disposed in between said flow            channel and said flow control channel to regulate fluid flow            through said flow channel, wherein said flow control valve            is deflectable into or retractable from said flow channel            upon which said flow control valve operates in response to            an actuation force applied to said flow control channel,            said flow control elastomeric segment when positioned in            said flow channel restricting fluid flow therethrough, and        -   (iv) a flow control channel actuation system operatively            interconnected to said flow control channel for applying an            actuation force to said flow control channel; and    -   (b) connecting the fluid delivery system to a chromatography        column having a column inlet and a column outlet such that the        column inlet is in fluid communication with the flow channel        outlet, wherein the chromatography column comprises a stationary        phase which is capable of separating at least a portion of the        analyte in the fluid.

In addition, methods for producing the microfluidic chromatographyapparatus can further include

-   -   (a) microfabricating the chromatography column which comprises a        chromatography channel having an inner surface which comprises a        functional group; and    -   (b) attaching a stationary phase compound to at least a portion        of the inner surface by reacting the stationary phase compound        with the functional group under conditions sufficient to form a        covalent bond between the functional group and the stationary        phase compound.

In one particular embodiment, the functional group is silane.

In another embodiment, the stationary phase compound is 1-octadecene.

The method can also include microfabricating a rotary channel describedabove.

Yet another aspect of the present invention provides a method forseparating an analyte from a sample fluid comprising:

-   -   (a) introducing the sample fluid into a microfluidic        chromatography apparatus described above, and    -   (b) eluting the sample fluid through the chromatography column        with an eluent to separate at least a portion of the analyte.

In one particular embodiment, fluid flow through the chromatographycolumn is achieved by a peristaltic pump action created by actuating oneor more of the flow control valves.

When the chromatography column comprises a microfabricated rotarychannel, the method can further include:

-   -   introducing at least a portion of the sample fluid into the        rotary channel;    -   closing the rotary inlet and the rotary outlet control valves by        actuating the rotary inlet and the rotary outlet control valves;    -   transporting the sample fluid through the rotary channel by        actuating one or more of the rotary pump valves until at least a        portion of the analyte is adsorbed onto the stationary phase;    -   opening the rotary inlet and rotary outlet control channels;    -   introducing a first eluent through the rotary inlet channel and        removing the resulting mixture through the rotary outlet        channel, whereby substantially all of the sample fluid is        removed from the rotary channel and at least about 95% of the        adsorbed analyte remains adsorbed onto the stationary phase; and    -   introducing a second eluent, which is capable of removing the        analyte from the stationary phase, through the rotary inlet        channel and removing the resulting mixture through the rotary        outlet channel, whereby substantially all of the adsorbed        analyte is removed from the rotary channel.

Such rotary channel chromatography column is particularly useful inseparating a large molecules such as proteins and oligonucleotides. Inone particular embodiment, the analyte is a protein having a molecularweight of at least about 1000 g/mol. Suitable stationary phases forproteins and oligonucleotides are well known to one skilled in the art.For example, proteins in aqueous solution can be separated using C-18alkyl as the stationary phase. In this manner, the first eluent isselected from the group consisting of water and an aqueous buffersolution, which removes the sample fluid but substantially leaves theadsorbed proteins bound to the solid phase. By using a second eluentwhich comprises an organic solvent selected from the group consisting ofan alcohol, acetonitrile, dimethylformamide, and mixtures thereof, onecan then remove the protein from the stationary phase. The second eluentcan also be a mixture of the organic solvent and water or an aqueousbuffer solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a front view illustration of an open capillary comprising acovalently bound surface modifying compound as a stationary phase.

FIG. 1B is a side cross-sectional view illustration of an open capillarycomprising a covalently bound surface modifying compound as a stationaryphase.

FIG. 2A is a front view illustration of a packed capillary comprising acovalently bound surface modifying compound as a stationary phase.

FIG. 2B is a side cross-sectional view illustration of a packedcapillary comprising a covalently bound surface modifying compound as astationary phase.

FIG. 3 is an illustration of a microfluidic chromatography apparatuscomprising an open capillary.

FIG. 4 is an illustration of a microfluidic chromatography apparatuscomprising an a packed capillary.

FIG. 5 is an illustration of a microfluidic chromatography apparatuscomprising both open capillary and packed capillary portions.

FIG. 6 is an illustration of a microfluidic device operativelyinterconnected to a capillary tube.

FIG. 7A is a front view of the first elastic layer integrated with achromatography column.

FIG. 7B is a side cross-sectional view showing the first elastic layerfitted with a chromatography column with dead volume in between thefluid channel and the chromatography column.

FIG. 7C is a side cross-sectional view showing the first elastic layerfitted with a chromatography column having a tapered fitting end whichreduces the amount of dead volume.

FIG. 8A is a perspective view of the first elastic layer having arectangular cross-section fluid flow channel.

FIG. 8B is a cut-away view along 1-1′ of FIG. 41A showing a taperedportion of fluid flow channel which is designed to reduce the amount ofdead volume between the chromatography column and the fluid flowchannel.

FIG. 8C is a front view of the first elastic layer fitted with achromatography column illustrating a possible gap formation between thechromatography column and the fluid flow channel.

FIG. 9 is another embodiment of attaching a chromatography column to amicrofluidic device of the present invention through the Z-axis.

FIG. 10 is an illustration of a rotary pump chromatography column of amicrofluidic device of the present invention.

FIG. 11 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 12 is an illustration of a second elastomeric layer formed on topof a micromachined mold.

FIG. 13 is an illustration of the elastomeric layer of FIG. 12 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 14 is an illustration corresponding to FIG. 13, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 15 is an illustration corresponding to FIG. 14, but showing thefirst and second elastomeric layers bonded together.

FIG. 16 is an illustration corresponding to FIG. 15, but showing thefirst micromachine mold removed and a planar substrate positioned in itsplace.

FIG. 17A is an illustration corresponding to FIG. 16, but showing theelastomeric structure sealed onto the planar substrate.

FIGS. 17B is a front sectional view corresponding to FIG. 17A, showingan open flow channel.

FIG. 17C corresponds to FIG. 17A, but shows a first flow channel closedby pressurization in second flow channel.

FIG. 18 is an illustration of a first elastomeric layer deposited on aplanar substrate.

FIG. 19 is an illustration showing a first sacrificial layer depositedon top of the first elastomeric layer of FIG. 18.

FIG. 20 is an illustration showing the system of FIG. 19, but with aportion of the first sacrificial layer removed, leaving only a firstline of sacrificial layer.

FIG. 21 is an illustration showing a second elastomeric layer applied ontop of the first elastomeric layer over the first line of sacrificiallayer of FIG. 20, thereby encasing the sacrificial layer between thefirst and second elastomeric layers.

FIG. 22 corresponds to FIG. 21, but shows the integrated monolithicstructure produced after the first and second elastomer layers have beenbonded together.

FIG. 23 is an illustration showing a second sacrificial layer depositedon top of the integral elastomeric structure of FIG. 22.

FIG. 24 is an illustration showing the system of FIG. 23, but with aportion of the second sacrificial layer removed, leaving only a secondline of sacrificial layer.

FIG. 25 is an illustration showing a third elastomer layer applied ontop of the second elastomeric layer and over the second line ofsacrificial layer of FIG. 24, thereby encapsulating the second line ofsacrificial layer between the elastomeric structure of FIG. 22 and thethird elastomeric layer.

FIG. 26 corresponds to FIG. 25, but shows the third elastomeric layercured so as to be bonded to the monolithic structure composed of thepreviously bonded first and second elastomer layers.

FIG. 27 corresponds to FIG. 26, but shows the first and second lines ofsacrificial layer removed so as to provide two perpendicularoverlapping, but not intersecting, flow channels passing through theintegrated elastomeric structure.

FIG. 28 is an illustration showing the system of FIG. 27, but with theplanar substrate thereunder removed.

FIGS. 29A is a front sectional view corresponding to FIG. 17A, showingan open flow channel.

FIG. 29B corresponds to FIG. 17A, but shows a first flow channel closedby pressurization in second flow channel.

FIGS. 30 a and 30 b illustrate valve opening vs. applied pressure forvarious flow channel dimensions.

FIG. 31A is a top schematic view of an on/off valve.

FIG. 31B is a sectional elevation view along line 23B-23B in FIG. 31A

FIG. 32A is a top schematic view of a peristaltic pumping system.

FIG. 32B is a sectional elevation view along line 24B-24B in FIG. 32A

FIG. 33 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIGS.32A and 32B.

DEFINITION

The terms “reactive polymerizable functional group”, “polymerizablefunctional group”, and “functional group” are used interchangeablyherein and refer to a functional group present in the monomeric orprepolymer or pre-crosslinked polymer unit(s) of the polymer which reactto form a polymer. It should be appreciated that the reactive functionalgroup refers to a functional group that is inherently present in thepolymer without any additional treatment, e.g., activation, of thepolymer. Exemplary reactive functional groups include, but are notlimited to, silane, alkene, isocyanate, epoxide, hydroxyl, and the like.

“Complimentary reactive polymerizable functional group” refers to afunctional group present in each polymer component, i.e., monomer orprepolymer or pre-crosslinked polymer, that react with each other toform a polymer.

“Active functional group” of a stationary phase compound refers to afunctional group present in the stationary phase compound which reactswith the functional group of the polymer to form a covalent bond.Exemplary active functional groups include, but are not limited to,hydroxy, alkene, silane, epoxide, isocyanate, and the like.

“Off ratio polymer” refers to a polymer which is produced from acombination of two or more monomeric or prepolymer or pre-crosslinkedpolymer units in which at least one monomeric component is present inexcess of the other component(s).

“Biocompatible polymer” refers to a polymer which when exposed to a celldoes not significantly change the cell morphology, cell and proteinactivity, and other cellular functions.

“Distribution equilibrium” refers to the ratio of the amount of asubstrate bound, i.e., adhered, to the stationary phase of the column orthe fluid flow channel and the amount of the substrate dissolved in thesolution.

“Rotary” refers to a configuration of a channel which allows circulationof a fluid within a confined region or section of the channel. Suchconfiguration can be a polygon, such as rectangle, hexagon, octagon, andthe like; or, preferably, an ellipse or a circle.

The terms “microfabricated flow channel,” “flow channel,” “fluidchannel,” and “fluid flow channel” are used interchangeably herein andrefer to a channel in a microfluidic device in which a fluid, such asgas or, preferably, liquid, can flow through.

The terms “chromatography column” and “column” are used interchangeablyherein and refers to a device or an apparatus which comprises astationary phase that is capable of separating at least a portion of ananalyte in a fluid.

The term “valve” unless otherwise indicted refers to a configuration inwhich two channels are separated by an elastomeric segment that can bedeflected into or retracted from one of the channels in response to anactuation force applied to the other channel.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a microfluidicchromatography apparatus, a method for producing the same, and a methodfor using the same. Preferably, the microfluidic chromatographyapparatus of the present invention comprises a microfabricated fluiddelivery system and a chromatography column, preferably an OTLC column,PCLC column, or combinations thereof. The chromatography column can bean integral part of the microfabricated fluid delivery system and assuch it can be microfabricated within the microfabricated fluid deliverysystem. Preferably, the chromatography column can be fabricatedseparately and integrated into the microfabricated fluid deliverysystem. The microfabricated fluid delivery system of the presentinvention is capable of delivering the fluid at a flow rate of 100μL/min or less, preferably 10 μL/min or less, and more preferably 1μL/min or less.

The microfabricated fluid delivery system of the present invention isproduced from a polymer, preferably an elastomeric polymer. In oneparticular embodiment of the present invention, the microfabricatedfluid delivery system is typically constructed at least in part fromelastomeric materials and constructed by single and multilayer softlithography (MLSL) techniques and/or sacrificial-layer encapsulationmethods. See, for example, Unger et al. (2000) Science 288:113-116, U.S.patent application Ser. No. 09/605,520, filed Jun. 27, 2000, and PCTPublication No. WO 01/01025, all of which are incorporated by referenceherein in their entirety. Thus, the microfluidic devices of the presentinvention comprise a microfabricated fluid flow channel (i.e., flowchannel or fluid flow channel).

In one aspect, the chromatography column itself is also produced from amicrofluidic device by modifying the inner surface of the chromatographychannel. In one embodiment, the chromatography column comprises astationary phase which is covalently attached to the inner surfacace ofthe chromatography channel. Preferably, the stationary phase iscovalently bonded to the inner surface. The stationary phase modifiesthe inner surface characteristics of the chromatography channel suchthat it is capable of separating an analyte from a sample fluid.Depending on the particular stationary phase (i.e., surface modifyingcompound) used, the chromatography column can be used in reverse phase,normal phase, hydrophobic interaction, affinity, etc., chromatography.Thus, a variety of chromatography columns can be fabricated by selectingan appropriate stationary phase compound. Typically, the stationaryphase is selected based on a particular analyte to be separated from thesample fluid.

Conventional surface coated polymers require activating the polymersurface before forming a bond with a surface modifying compound (e.g.,stationary phase compound). In contrast, microfluidic chromatographycolumn devices of the present invention are preferably made frompolymers such that the resulting chromatography column devices comprisea functional group within the inner surface of the chromatographychannel. Thus, in one embodiment, microfluidic chromatography columndevices of the present invention do not require a separate inner surfaceactivation step for covalent bonding the stationary phase compound. Thestationary phase is covalently bonded to the chromatography channel byforming a covalent bond between an active functional group of thestationary phase compound and the functional group of the polymer. Asstated above, the functional group is inherently present on the polymersurface prior to contacting it with a stationary phase compound, andtherefore a separate activation step is not required. The amount offunctional group on the inner surface of the chromatography channelshould be sufficient enough such that a useful chromatography column isformed by reacting with a stationary phase compound. Typically on theaverage, polymers that are used to produce microfluidic chromatographycolumn devices of the present invention comprise at least one functionalgroup per 10,000 monomeric units on the inner surface of thechromatography channel. Preferably, polymers of the present inventioncomprise at least one functional group per 1,000 monomeric units on theinner surface. And more preferably, polymers of the present inventioncomprise at least one functional group per 100 monomeric units on theinner surface.

In one aspect of the present invention, the microfluidic devices (e.g.,fluid delivery devices and/or chromatography column devices) areproduced from polymers by combining two or more different polymercomponents (e.g., monomers) in which each polymer component includes acomplimentary reactive functional group. The ratio of each component isselected such that there is an excess of at least one component toprovide unreacted function group within the polymer surface, includingany inner surface. Preferably, polymers of the present inventioncomprise at least one polymerizable functional group per 10,000monomeric units within the polymer bulk matrix. More preferably,polymers of the present invention comprise at least one polymerizablefunctional group per 1,000 monomeric units within the polymer bulkmatrix. And most preferably, polymers of the present invention compriseat least one polymerizable functional group per 100 monomeric unitswithin the polymer bulk matrix.

Stationary Phase Compound

It has been found by the present inventors that by using “off ratiopolymers” with the quantity of unreacted functional group describedabove, a stationary phase compound with an appropriate activate functiongroup can be covalently attached to the polymer surface without the needfor a polymer surface activation step. Such off ratio polymers aredisclosed in the commonly assigned co-pending U.S. Provisional PatentApplication Ser. No. 60/281,929, entitled “Polymer SurfaceModification,” filed on Apr. 6, 2001 which is incorporated herein byreference in its entirety. However, it should be appreciated thatdepending on the particular polymer used, the process for producingmicrofluidic devices of the present invention can also includeactivating the polymer surface for covalent linkage with a stationaryphase compound. For example, such a surface activation is particularlydesirable where the column is fabricated from a different material thanthe fluid delivery system.

Stationary phase compounds are selected such that the resulting columnis capable of separating at least a portion of the desired analyte fromthe sample fluid. Suitable stationary phase compounds for a particularanalyte are well known to one skilled in the art. For example, usefulstationary phase compounds include, but are not limited to,1-octadecanol, 1-octadecene, octadecylsilane, octadecyltrichlorosilane,octadecyl isocyanate, trioctedecylsilane, etc. for C₁₈ grafting (e.g.,stationary phase for C₁₈ reverse phase LC), and corresponding compoundsfor C₈, C₄ or C₂ stationary phase.

The stationary phase compounds can be attached to the inner surface ofthe column by contacting the stationary phase compound to the innersurface under conditions sufficient to produce a covalent bond. Forexample, attachment of 1-octadecene to a polymer comprising a silanefunctional group (e.g., Si—H) can be achieved by contacting, e.g,immersing, spraying, or coating, the polymer with 1-octadecene whichhave a terminal olefin group. The silane group reacts with the olefingroup to form an alkyl-silane bond to produce a C-18 stationary phasecolumn. In one particular embodiment of the present invention, thestationary phase compound is useful in forming an OTLC column, PCLCcolumn, or mixtures thereof.

Typically, at least about 1 equiv. of the stationary phase compound isused, preferably at least about 10 equiv., and more preferably at leastabout 100 equiv. As used herein, the equiv. of the stationary phasecompound refers to the equiv. amount of the functional group of thestationary phase compound relative to the theoretical amount of thefunctional group present on the polymer surface to be treated. Use of anexcess amount of the stationary phase compound ensures a substantiallycomplete and a relatively fast surface coating. Any excess stationaryphase compound that is not covalently attached to the inner surface isthen removed from the column.

When forming a covalent bond with the functional group of the polymer,the stationary phase compound can be in the form of a solution in aninert solvent. Or if the stationary phase compound is a liquid or a gas,it can be used directed without any solvent. When the stationary phasecompound is in a solution, the solvent used is, preferably, inert to thereaction conditions. Suitable inert solvents for a particular reactivefunctional group are well known to one of ordinary skill in the art. Forexample, suitable inert solvents for a silane reactive functional groupinclude hydrocarbons, ethyl ether, tetrahydrofuran, dimethoxyethane(DME), dimethyl formaldehyde, chloroform, dichloromethane, toluene,xylene, and the like.

The reaction temperature between the intrinsic functional group of thepolymer and the stationary phase compound depends on a variety offactors including, the stability of the polymer at a particulartemperature, concentration and reactivity of the function groups, thestability of the covalent bond that is formed, etc. For example, inreacting 1-octadecene to a polymer comprising a silane group, thereaction temperature is typically from about 20° C. to about 200° C.,preferably from about 50° C. to about 150° C., and more preferably fromabout 70° C. to about 90° C.

The reaction time also depends on a variety of factors such asconcentration of the functional group and the stationary phase compound,reaction temperature, reactivity of the functional group and thestationary phase compound, etc. For reacting 1-octadecene to a polymercomprising a silane group, the reaction time is typically from about 2 hto about 60 h, preferably from about 8 h to about 48 h, and morepreferably from about 12 h to about 24 h.

Basic Features of the Microfluidic Fluid Delivery System

In one particular aspect of the present invention, microfluidic devicesare constructed at least in part from elastomeric materials. Typically,the microfluidic devices are constructed by single and multilayer softlithography (MLSL) techniques and/or sacrificial-layer encapsulationmethods as disclosed in the above incorporated U.S. patent applicationSer. No. 09/605,520, filed Jun. 27, 2000, PCT Publication No. WO01/01025, and Unger et al. (2000) Science 288:113-116.

Microfluidic devices of the present invention comprise a microfabricatedflow channel. In addition, microfluidic devices of the present inventioncan optionally further comprise a variety of plumbing components (e.g.,pumps, valves, and connecting channels) for flowing fluids such asreagents, solvents, and samples. The microfluidic devices can alsocomprise an array of reservoirs for storing reaction reagents (e.g.,solvents, samples, eluents, and other reagents can each be stored in adifferent reservoir).

The microfluidic devices of the present invention have a basic “flowchannel” structure. The term “flow channel”, “fluid channel”, or“microfabricated flow channel” refers to a channel in which a fluid,such as gas or, preferably, liquid, can flow through. The flow channelscan also be actuated to function as the plumbing components (e.g.,micro-pumps, micro-valves, or connecting channels) of the microfluidicdevices.

In some applications, microfabricated flow channels are cast on a chip(e.g., a elastomeric chip). Fluid channels are formed by bonding thechip to a flat substrate (e.g., a glass cover slip or another polymer)which seals the channel. Thus, one side of the synthesis channel isprovided by the flat substrate. Typically, the stationary phase compoundis attached to the inner surface of the polymer within thechromatography channel. However, when the flow channel is formed byattaching the polymer to a solid substrate, such as glass, the innersurface of the flow channel comprises a polymer portion and a solidsubstrate portion. In essence, the flow channel is formed on aninterface between the polymer and the solid substrate. In oneembodiment, the solid stationary phase is covalently bonded to the solidsubstrate. The surface of the solid substrate (i.e., inner surface ofthe solid substrate portion of the flow channel) can be etched ormodified to include arrays of pillars, columns, pyramides, etc. toincrease the surface area of the chromatography column. Such surfacemodifications of a solid substrate can be readily achieved usingstandard wafer/glass process steps. In another embodiment, the polymerportion of the flow channel inner wall is coated with a surfacemodifying compound to reduce non-specific bonding (NBS) of the analyte,i.e., passivated. Alternatively, the solid substrate portion can bepassivated and the polymer portion can be covalently bonded to thestationary phase compound.

The plumbing components can be microfabricated as described in the aboveincorporated references. For example, the microfluidic devices cancontain an integrated flow cell (i.e., reservoir) in which a pluralityof fluid channels are present, and fluidic components (such asmicro-pumps, micro-valves, and connecting channels) for controlling theflow of the reagents into and out of the flow cell. Alternatively, themicrofluidic devices of the present invention can utilize other plumbingdevices. See for example, Zdeblick et al., A MicrominiatureElectric-to-Fluidic Valve, Proceedings of the 4th InternationalConference on Solid State Transducers and Actuators, 1987; Shoji et al.,Smallest Dead Volume Microvalves for Integrated Chemical AnalyzingSystems, Proceedings of Transducers '91, San Francisco, 1991; andVieider et al., A Pneumatically Actuated Micro Valve with a SiliconRubber Membrane for Integration with Fluid Handling Systems, Proceedingsof Transducers '95, Stockholm, 1995, all of which are incorporatedherein by reference in their entirety.

At least some of the components of the microfluidic devices aremicrofabricated. Employment of microfabricated fluid channels and/ormicrofabricated plumbing components significantly reduce the dead volumeand decrease the amount of time needed to exchange reagents, which inturn increase the throughput. Microfabrication refers to featuredimensions on the micron level, with at least one dimension of themicrofabricated structure being less than 1000 μm. In some microfluidicdevices, only the fluid channels are microfabricated. In somemicrofluidic devices, in addition to the fluid channels, the valves,pumps, and connecting channels are also microfabricated. Unlessotherwise specified, the discussion below of microfabrication isapplicable to production of all microfabricated components of themicrofluidic devices (e.g., the fluid channels, valves, pumps, andconnecting channels).

As discussed in detail below, various materials can be used to producethe microfluidic devices. Preferably, elastomeric materials are used.Thus, in some microfluidic devices, the integrated (i.e., monolithic)microstructures are made out of various layers of elastomer bondedtogether. By bonding these various elastomeric layers together, therecesses extending along the various elastomeric layers form fluidchannels through the resulting monolithic, integral elastomericstructure.

In general, the microfabricated structures (e.g., fluid channels, pumps,valves, and connecting channels) have widths of about 0.01 to 1000microns, and a width-to-depth ratios of between 0.1:1 to 100:1.Preferably, the width is in the range of 10 to 200 microns, awidth-to-depth ratio of 3:1 to 15:1.

Microfluidic Chromatogaphy

Carrying out chemical or biochemical analyses, syntheses orpreparations, even at the simplest levels, requires one to perform alarge number of separate manipulations on the material components ofthat analysis, synthesis or preparation, including measuring,aliquoting, transferring, diluting, concentrating, separating,detecting, etc. In this respect, microfluidic devices of the presentinvention are particularly useful in performing these manipulations,particularly in separation of the analyte, in a microscale level.

In order to manipulate reagents (e.g., samples, eluents, etc.) withinthe microfabricate devices described herein, the overall microfabricatedevices of the present invention typically include a pumps, valves,various channels, and/or chambers. Pumps and valves generally aredesigned to controls the movement and direction of fluids within theflow channel. Generally, pump and valve systems employ pressure or otherknown actuation systems to affect fluid movement and fluid flowdirection. Preferably, the microfluidic devices of the present inventioncomprise the pump and valve systems, which are described in detailbelow. Other fluid movement and direction controls for microfluidicdevices are known in the art, including mechanical pumps and valves andelectroosmotic fluid direction systems. Such fluid movement anddirection controls are contemplated to be within the scope of thepresent invention. Electroosmotic fluid direction systems andcontrollers are well known and described in detail, for example, in U.S.Pat. No. 5,779,868, which is incorporated herein by reference in itsentirety.

The present invention will be described with regard to the accompanyingdrawings which assist in illustrating various features of the invention.In this regard, the present invention generally relates to microfluidicchromatography apparatuses. In one aspect of the present invention,microfluidic devices of the present invention comprise an OTLC column,PCLC column, or combinations thereof. It should be appreciated that thedrawings are provided for the purpose of illustrating the practice ofthe present invention and do not constitute limitations on the scopethereof.

Referring to FIGS. 1A and 1B, OTLC column 110 comprises a flow channel96 having an inner surface 100 and a stationary phase 104 whichcomprises a stationary phase compound covalently bonded to the innersurface 100. The stationary phase 104 is capable of separating ananalyte in a solution, and as such the selection of a particularstationary phase compound depends on the particular analyte to beseparated.

FIGS. 2A and 2B illustrate PCLC 120 which comprises a chromatographychannel 124 packed with a solid adsorbent 128. The solid adsorbent 128can comprise a solid polymer, e.g., plastic, glass, and other polymers,which is coated or, preferably, covalently bonded to a stationary phasecompound. Alternatively, the solid adsorbent 128 can be a conventionalchromatography adsorbent such as paper, cellulose, starch, sugars,magnesium silicate, calcium sulfate, silicic acid, silica gel, florisil,magnesium oxide, aluminum oxide (alumina), activated charcoal, and thelike. It should be appreciated these conventional chromatographyadsorbents are not coated or covalently bonded to a separate stationaryphase compound. In these adsorbents, their surface contains moieties,e.g., functional group such as hydroxy groups, that effect separation ofthe analyte. In conventional PCLC columns, the solid adsorbent 128 istypically not bound to the inner surface of the chromatography channel124, which can result in the solid adsorbent 128 leaking out of thechromatography channel 124 during its operation. Leakage of the solidadsorbent 128 from the chromatography channel 124 can be prevented bytapering the outlet portion of the chromatography channel 124.

Preferably, the solid adsorbent 128 in PCLC 120 is an integral part ofthe microfluidic chromatography channel and is covalently bonded to astationary phase compound, i.e., the solid adsorbent 128 comprises aplurality of protuberances that are present on the inner surface of thechromatography channel. Thus, the solid adsorbent 128 comprises a samepolymeric material as the microfluidic chromatography column itselfwhich is covalently bonded to a stationary phase compound. Such achromatography channel can be readily fabricated by using a mold havinga non-smooth surface, e.g., mold having protuberances or depressions, orother suitable polymer fabrication techniques known to one skilled inthe art. Thus, in this embodiment, the chromatography channel 124 istechnically not “packed” but is comprised of a plurality of innersurface protuberances within the inner surface of the chromatographychannel 124. One of the advantages for having a plurality ofprotuberances within the inner surface is an increase in the totalsurface area of the inner surface of the chromatography channel 124,which results in a longer net effective column length and a higher neteffective column plate number.

It should be appreciated that the microfluidic devices of the presentinvention can comprise a plurality of OTLC columns, PCLC columns, orcombinations thereof. Such plurality of columns can be arranged inseries, see FIG. 5, to provide separation of a number of analytes withina single sample fluid. They can also be arranged in parallel (not shown)to provide separation of a number of solutions in a single microfluidicdevice. Or the columns can be arranged in both series and parallelmanner (not shown) to allow separation of a number of analytes from anumber of sample fluids on a singe microfluidic device.

FIGS. 3, 4 and 5 correspond to microfluidic devices comprising an OTLCcolumn, PCLC column and a combination of OTLC and PCLC columns,respectively. A sample (neat or in a solution) is introduced through theinlets 108, 138 and 148 of FIGS. 3, 4 and 5, respectively. A pump andvalve system (not shown) moves the sample through the columns 110, 120and 140 (shown in phantom). An eluent can be introduced through the sameinlet 108, 138 and 148 or the microfluidic devices can further comprisean eluent inlet and an eluent reservoir (not shown) that isinterconnected to the columns 110, 120, and 140 near the inlets 108, 138and 148, respectively. A pump and valve system (not shown) can be usedto control the flow of eluent through the columns 110, 120 and 140. Asthe solutes (i.e., analyte) passes down the columns 110, 120 and 140 akind of distribution equilibrium is established between the stationaryphase (i.e., adsorbent material or surface modifying compound) and thesolvent. The distribution equilibrium refers to the equilibriumestablished between the solute being adsorbed onto the stationary phaseand the amount of solute dissolved in the solvent. Such distributionequilibrium depends on the strength of interaction between the soluteand the stationary phase, and the solubility of the solute in a givensolvent. Useful solvents for a particular stationary phase and analyteare well known to one skilled in the art or can be readily determinedwithout undue experimentation. Typically, different solutes havedifferent distribution equilibrium. Therefore, different solutes willmove down the columns 110, 120 and 140 at differing rates depending ontheir relative affinity for the adsorbent (i.e., stationary phase) onone hand and for the solvent on the other. As the components of themixture (i.e., analytes) are separated, they begin to form moving bandsor zones. Preferably, the length of columns 110, 120 and 140 are chosensuch that the bands are separated from one another, leaving gaps of puresolvent in between. The outlets 114, 134 and 144 can be interconnectedto a detector, such as gas chromatography, IR, UV/VIS, or MassSpectrometer, for analyzing the separated solute. Alternatively, theoutlets 114, 134 and 144 can be interconnected to another microfluiddevice which can further manipulate the separated sample, e.g., PCRamplification of nucleotides.

In one aspect, the microfluidic chromatography column device and themicrofabricated fluid delivery system are fabricated separately andintegrated with each other such that one microfluidic device serves as afluid delivery or injection system and the other is used aschromatography column. Advantages of this aspect of the inventioninclude the capability of using the microfabricated fluid deliverysystems with a variety of different chromatography columns andinterchangeability of chromatography columns depending on the need. Onesuch embodiment is illustrated in FIG. 6, where a chromatography column200 is operatively interconnected to a microfluidic device 204. Thisallows use of the microfluidic device 204 with a various chromatographycolumns and applications. The microfluidic devices 204 can comprise avariety of components, such as a component for sample concentration,sample dilution, sample preparation components, etc.

In one aspect of the present invention, commercially availablechromatography columns can be purchased and used in conjunction with themicrofluidic device 204. Such OTLC and PCLC columns can be readilyproduced without difficulties. Referring again to FIG. 6, thechromatography column 200 can be interconnected to the microfluidicdevice 204 simply by inserting the column 200 in to the flow channel(not shown) of the microfluidic device 200. In this manner, the column200 sits within the flow channel (not shown) and extends beyond the edgeof the microfluidic device 204. The length of the column 200 depends ona variety of factors including, but not limited to, the amount of columnlength required to separate the analyte from the sample.

As shown in FIGS. 7A-7C, the column 200 can be sealed within twoportions (i.e., layers 208 and 212) of the microfluidic device 204. Itcan be sealed either directly by baking together the two portions ofpartially cured elastomers or by incorporation of uncured elastomer(e.g., RTV, discussed in detail below) during the final bake (i.e.,curing) stage. In this arrangement, fluids are designed to flow in themiddle of the two portions (i.e., top portion 208 and bottom portion212) of the microfluidic device 204. The alignment of the column 200between the two portions and its juxtaposition with the fluid channel216 can create a partial occlusion of the capillary when the column 200is centered between the layers. Better alignments can be achieved bycreating an offset in the depths (i.e., height) of two portions of thechannels between which the capillary is fitted. For example, if thedepth of the lower portion is 5 microns less than the upper portion ofthe polymer, a column with a ten-micron internal diameter can beaccommodated without a significant offset.

The portion of flow channel 216 that becomes integrated with the column200 is configured such that the fluid sample flows directly from themicrofluidic device 204 to the column 200. And the column 200 can befurther interconnected to a sample analytical device, a collectiondevice, or another microfluidic device(s) for further manipulation ofthe separated analyte (not shown). Additional features patterned inpolymer may be necessary to reduce potential dead volume 220 at thejunction between the column 200 and the flow channel 216. Alternatively,the amount of dead volume can be reduced by using a tapered column 200as shown in FIG. 7C. Typical, dimensions of the OTLC or PCLC columnsthat can be accommodated in microfluidic devices of the presentinvention include, but are not limited to, columns with internaldiameters of from about 500 μm to about 2 μm and outer diameters of fromabout 1000 μm to about 10 μm.

The column 200 can be sealed within the microfluidic device 204, by avariety of processes. For example, the column 200 can be sealed duringbaking together of the two portions of the elastic layers.Alternatively, as shown in FIGS. 8A-8C, the column 200 is ‘push-fit’into the microfluidic device 204 having a slightly smaller flow channeldiameter than the outer diameter of the column 200, thereby creating aninstant seal. The dimensions of the push-fit envelope are chosen toaccommodate the diameter of the column 200. For example, an envelope ofabout 200 μm width and about 15 μm in height has a perimeter of 430microns. A column 200 with 100 μm outer diameter has a circumference of314 μm. The seal can be further secured by incorporation of uncuredelastomer (e.g., RTV) in the envelope between the two portions (e.g.,areas 224A and 224B in FIG. 8C). As shown in FIG. 9, push fitting canalso be used to incorporate a column 200 that fits into the device inthe ‘Z’ plane. One major advantage of push fitting is that column 200can be easily interchanged if clogging occurs.

Chromatography separation results depend on many factors including, butnot limited to, the adsorbent (i.e., stationary phase compound) chosen,polarity of the solvent, size of the column (both length and diameter)relative to the amount of material to be chromatographed, and the rateof elution. Columns shown in FIGS. 3, 4 and 5 are single pass columns,i.e., samples and solutions travel through the column only once duringoperation. Thus, in some cases a long column or multiple columnsarranged in series may be required to separate the sample effectively.This is particularly true when the sample has a relatively lowdistribution equilibrium between the stationary phase and the solvent.In other cases, the sample can bind tightly to the adsorbent materialand may require a different solvent to elute the sample from theadsorbent. For example, proteins/peptides with molecular weight ofgreater than 1000 in aqueous medium bind tightly to C-18 alkylstationary phase. This bonding is so strong that it is difficult toeffectively remove the protein from the stationary phase using water.Typically an organic eluent, such as acetonitrile, alchohol (e.g.,methanol, ethanol, or isopropanol), other relatively polar organicsolvents (e.g., DMF), or mixtures thereof, is used as an eluent toremove the protein from the stationary phase.

Present inventors have found that this difference in the distributionequilibrium of samples, e.g., proteins, in different solvents can beused advantageously with microfluidic devices of the present inventionin some sample separations. One such configuration is illustrated inFIG. 10 which will be described in reference to separating proteins.However, it should be appreciated that other compounds having a similardistribution equilibrium difference in different solvents can beseparated using the principle disclosed herein.

The microfluidic device of FIG. 10 comprises a rotary flow channel 300which has an inlet 304 and an outlet 308. The flow channel 300 iscovalently bonded to a stationary phase compound, such as C-18 alkyl,that binds strongly to proteins in aqueous solution. An aqueous proteinsolution is introduced into the rotary flow channel 300 by opening thecontrol valves 312 and 316. If the volume of the sample is insufficientto completely fill the rotary flow channel 300, additional water can beadded through the inlet 304. Water can be introduced through the samesample port 320 or, as shown in FIG. 10, a separate solvent port 324 canbe present in the microfluidic devices. Optionally, the microfluidicdevices can further comprise an additional solvent port 328 forintroducing a second solvent which can be mixed with the first solventthat is introduced through the solvent port 324. Preferably, eachsolvent port has its own pump and control valve systems 332 and 336.

After the rotary flow channel 300 is filled with the aqueous proteinsolution, control valves 312 and 316 are actuated to maintain a closedsystem. The aqueous protein solution is then circulated through therotary flow channel using a pump comprised of control valves 340A-340Duntil substantially all the high molecular proteins are bound to theinner surface of the flow channel 300. The rotary flow channel 300 canbe flushed with water by opening the control valves 312 and 316 andintroducing additional water through the inlet 304 and removing thesolution through the outlet 308. The exiting solution can be connectedto other rotary flow channel(s) (not shown) to further separate othercompounds that may be present, discarded, collected, or sent to adetector system to identify the contents of the exiting solution. Atthis stage, high molecular proteins are bound to the inner surface ofthe rotary flow channel 300 and low molecular proteins and other polarcompounds have been removed from the rotary flow channel 300. To recoverthe bound protein, acetonitrile, methanol, ethanol or mixtures thereof,or an aqueous mixture of such solvent is introduced to the rotary flowchannel 300 through the inlet 312. Presence of organic solvent lowersthe distribution equilibrium between the stationary phase and thesolvent, i.e., the amount of protein in the solution is increased. Theorganic solution containing dissolved proteins can be collected,analyzed, or further manipulated as needed. Alternatively, afterintroducing the organic solvent, control valves 312 and 316 can beclosed and the solvent circulated through the rotary fluid channel 300prior to removing the solution from the rotary fluid channel 300. Thisallows dissolution of proteins in a small volume of the organic solvent.

Basic Methods of Microfabrication

Various methods can be used to produce the microfabricated components ofthe microfluidic devices of the present invention. Fabrication of themicrochannels, such as flow channels, valves, and pumps, can beperformed as described in the above incorporated references. In somemethods, e.g., FIGS. 11 to 17B, pre-cured elastomer layers are assembledand bonded to produce a flow channel. As illustrated in FIG. 11, a firstmicro-machined mold 10 is provided. Micro-machined mold 10 can befabricated by a number of conventional silicon processing methodsincluding, but not limited to, photolithography, ion-milling, andelectron beam lithography. The micro-machined mold 10 has a raised lineor protrusion 11 extending therealong. A first elastomeric layer 20 iscast on top of mold 10 such that a first recess 21 can be formed in thebottom surface of elastomeric layer 20, (recess 21 corresponding indimension to protrusion 11), as shown.

As can be seen in FIG. 12, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 can be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 13 and 14,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 forms a controlchannel 32.

Referring to FIG. 15, the separate first and second elastomeric layers20 and 22 (FIG. 14) are then bonded together to form an integrated(i.e., monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 16 and 17A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIG. 17A and 17B, whenelastomeric structure 24 has been sealed at its bottom surface to planarsubstrate 14, recess 21 forms a flow channel 30.

The present elastomeric structures can form a reversible hermetic sealwith nearly any smooth planar substrate. An advantage to forming a sealthis way is that the elastomeric structures can be peeled up, washed,and re-used. In some microfluidic devices, planar substrate 14 is glass.A further advantage of using glass is that glass is transparent,allowing optical interrogation of elastomer channels and reservoirs.Alternatively, the elastomeric structure can be bonded onto a flatelastomer layer by the same method as described above, forming apermanent and high-strength bond. This can prove advantageous whenhigher back pressures are used.

In some methods, microfabrication involves curing each layer ofelastomer “in place” (FIGS. 18 to 28). In these methods, fluid flow andcontrol channels are defined by first patterning sacrificial layer onthe surface of an elastomeric layer (or other substrate, which caninclude glass) leaving a raised line of sacrificial layer where achannel is desired. Next, a second layer of elastomer is added thereoverand a second sacrificial layer is patterned on the second layer ofelastomer leaving a raised line of sacrificial layer where a channel isdesired. A third layer of elastomer is deposited thereover. Finally, thesacrificial layer is removed by dissolving it out of the elastomer withan appropriate solvent, with the voids formed by removal of thesacrificial layer becoming the flow channels passing through thesubstrate, i.e., microfluidic device.

Referring first to FIG. 18, a planar substrate 40 is provided. A firstelastomeric layer 42 is then deposited and cured on top of planarsubstrate 40. Referring to FIG. 19, a first sacrificial layer 44A isthen deposited over the top of elastomeric layer 42. Referring to FIG.20, a portion of sacrificial layer 44A is removed such that only a firstline of sacrificial layer 44B remains as shown. Referring to FIG. 21, asecond elastomeric layer 46 is then deposited over the top of firstelastomeric layer 42 and over the first line of sacrificial layer 44B asshown, thereby encasing first line of sacrificial layer 44B betweenfirst elastomeric layer 42 and second elastomeric layer 46. Referring toFIG. 22, elastomeric layers 46 is then cured on layer 42 to bond thelayers together to form a monolithic elastomeric substrate 45.

Referring to FIG. 23, a second sacrificial layer 48A is then depositedover elastomeric structure 45. Referring to FIG. 24, a portion of secondsacrificial layer 48A is removed, leaving only a second sacrificiallayer 48B on top of elastomeric structure 45 as shown. Referring to FIG.25, a third elastomeric layer 50 is then deposited over the top ofelastomeric structure 45 (comprised of second elastomeric layer 42 andfirst line of sacrificial layer 44B) and second sacrificial layer 48B asshown, thereby encasing the second line of sacrificial layer 48B betweenelastomeric structure 45 and third elastomeric layer 50.

Referring to FIG. 26, third elastomeric layer 50 and elastomericstructure 45 (comprising first elastomeric layer 42 and secondelastomeric layer 46 bonded together) is then bonded together forming amonolithic elastomeric structure 47 having sacrificial layers 44B and48B passing therethrough as shown. Referring to FIG. 27, sacrificiallayers 44B and 48B are then removed (for example, by dissolving in asolvent) such that a flow channel 60 and a control channel 62 areprovided in their place, passing through elastomeric structure 47 asshown. And referring to FIG. 28, planar substrate 40 can be removed fromthe bottom of the integrated monolithic structure.

Microfabricated Polymers

Microfabricated refers to the size of features of a polymer fabricatedin accordance with an embodiment of the present invention. In general,variation in at least one dimension of microfabricated structures iscontrolled to the micron level, with at least one dimension beingmicroscopic (i.e. below 1000 μm). Microfabrication typically involvessemiconductor or MEMS fabrication techniques such as photolithographyand spincoating that are designed for to produce feature dimensions onthe microscopic level, with at least some of the dimension of themicrofabricated structure requiring a microscope to reasonablyresolve/image the structure.

In preferred aspects, channels (flow channels and controls channels) 30,32, 60 and 62 preferably have width-to-depth ratios of about 10:1. Anon-exclusive list of other ranges of width-to-depth ratios inaccordance with embodiments of the present invention is 0.1:1 to 100:1,more preferably 1:1 to 50:1, more preferably 2:1 to 20:1, and mostpreferably 3:1 to 15:1. In an exemplary aspect, flow channels 30, 32, 60and 62 have widths of about 1 to 1000 microns. A non-exclusive list ofother ranges of widths of channels in accordance with embodiments of thepresent invention is 0.01 to 1000 microns, more preferably 0.05 to 1000microns, more preferably 0.2 to 500 microns, more preferably 1 to 250microns, and most preferably 10 to 200 microns. Exemplary channel widthsinclude 0.1 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm,160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm,and 250 μm.

Channels 30, 32, 60, and 62 have depths of about 1 to 100 microns. Anon-exclusive list of other ranges of depths of channels in accordancewith embodiments of the present invention is 0.01 to 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250 microns, andmore preferably 1 to 100 microns, more preferably 2 to 20 microns, andmost preferably 5 to 10 microns. Exemplary channel depths includeincluding 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm,3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, and 250μm.

The channels are not limited to these specific dimension ranges andexamples given above, and can vary in width in order to affect themagnitude of force required to deflect the elastomeric segment.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

Elastomeric layer 22 can be cast thick for mechanical stability. In anexemplary embodiment, layer 22 is 50 microns to several centimetersthick, and more preferably approximately 4 mm thick. A non-exclusivelist of ranges of thickness of the elastomer layer in accordance withother embodiments of the present invention is between about 0.1 micronto 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100 microns to 10 mm.

Accordingly, elastomeric segment 25 of FIG. 17B separating flow channel30 and control channel 32 has a typical thickness of between about 0.01and 1000 microns, more preferably 0.05 to 500 microns, more preferably0.2 to 250, more preferably 1 to 100 microns, more preferably 2 to 50microns, and most preferably 5 to 40 microns. As such, the thickness ofelastomeric layer 22 is about 100 times the thickness of elastomericlayer 20. Exemplary elastomeric segment thicknesses include 0.01 μm,0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm,30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400μm, 500 μm, 750 μm, and 1000 μm

Similarly, first elastomeric layer 42 can have a preferred thicknessabout equal to that of elastomeric layer 20 or 22; second elastomericlayer 46 can have a preferred thickness about equal to that ofelastomeric layer 20; and third elastomeric layer 50 can have apreferred thickness about equal to that of elastomeric layer 22.

One particular aspect of the present invention provides microfluidicdevices comprising a microfabricated flow channel which is locatedwithin the polymer matrix and defines an inner surface. Optionally, themicrofluidic devices comprise a plurality of microfabricated flowchannels. The microfluidic devices can also have a plurality ofreservoirs for storing various reagents such as solutions, solvents,and/or samples. In addition, the microfluidic devices can have pumps andvalves for controlling flow of the reagents. The flow channel can alsohave a window to allow optical interrogation.

Use of microfluidic devices of the present invention reduces the samplesize and the amount of eluent needed as well as providing a sufficientlysmall flow rate for microscale chromatography processes, e.g., OTLC orPCLC.

Polymers of the present invention are preferably produced frompolymerization of at least two different components. These polymers arepreferably produced using an off ratio of each component. Exemplary offratio polymers which are useful in the present invention include, butare not limited to:

-   -   silicone polymers which can be produced from monomers comprising        a silane and an olefin reactive polymerizable functional groups,        e.g., GE's RV615, and Dow Corning's Sylgard 184, 182 186;    -   polyurethane polymers which can be produced from monomers        comprising a diisocyanate and an di-alcohol or di-amine reactive        polymerizable functional groups, e.g., Synair's 2612020, 261S111        and 261S333 or Uniroyal's Vibrathane 504;    -   polyisoprene, polybutadiene, polychloroprene which are        polymerized from diene monomers, and therefore have one double        bond per monomer when polymerized. This double bond on the        surface allows the covalent bonding of the stationary phase        compound to the polymer. The polymer rubber can then be        vulcanized to form a soft elastomer product.    -   styrene butadiene rubber which is produced from an olefin and a        diene reactive functional groups of styrene and butadiene,        respectively; The double bond presented in the pre-crosslinked        polymer allows the surface of the polymer to be modified.

Preferable, polymers of the present invention comprise off ratio polymerderived from at least two PDMS resins containing silane and olefinfunctional groups, respectively.

The amount of each component is selected such that the relative molarratio of the reactive functional group of one monomeric unit is presentin excess of the other(s). In this manner, a significant amount of thereactive functional group of the excess monomer remains unreacted withinthe polymer. Preferably, at least about 1% of the reactive functionalgroup of the excess monomer remains unreacted within the polymer, morepreferably at least about 6%, and most preferably at least about 30%.Alternatively, polymers of the present invention comprise one unreactedreactive functional group per about 10,000 monomeric units, preferablyper about 1,000 monomeric units, and more preferably per about 100monomeric units.

In one particular embodiment, the polymer is derived from twomonomer/prepolymer components. Preferably, the polymer is produced bycombining the respective monomer/prepolymer at a relative molar ratio offrom 1:10 to about 1:3, more preferably at a relative molar ratio offrom 1:5 to about 1:2, and most preferably at a relative molar ratio offrom 1:2 to about 1:1.1.

Other Suitable Polymer Materials

Allcock et al., Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials can be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa toabout 1 TPa, more preferably between about 10 Pa to about 100 GPa, morepreferably between about 20 Pa to about 1 GPa, more preferably betweenabout 50 Pa to about 10 MPa, and more preferably between about 100 Pa toabout 1 MPa are useful in accordance with the present invention,although elastomeric materials having a Young's modulus outside of theseranges could also be utilized depending upon the needs of a particularapplication.

The systems of the present invention can be fabricated from a widevariety of elastomers, preferably off ratio polymers. For example,elastomeric layers 20, 22, 42, 46 and 50 can preferably be fabricatedfrom silicone rubber. In some applications, microstructures of thepresent systems are fabricated from an elastomeric polymer such as GERTV615 (formulation), a vinyl-silane crosslinked (type) siliconeelastomer (family). An important requirement for the preferred method offabrication is the ability to produce a polymer with unreacted reactivefunctional group. More preferably, the fabrication process produceslayers of elastomers which can be bonded together. In the case ofmultilayer soft lithography, layers of elastomer are cured separatelyand then bonded together. This scheme requires that cured layers possesssufficient reactivity to bond together. Either the layers can be of thesame type, and are capable of bonding to themselves, or they can be oftwo different types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microstructures. Variations in the materialsused most likely are driven by the need for particular materialproperties, e.g., stiffness, gas permeability, or temperature stability.

Common elastomeric polymers include, but are not limited to,polyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones. Thefollowing is a non-exclusive list of elastomeric materials which can beutilized in connection with the present invention: polyisoprene,polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and siliconepolymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)(nitrile rubber), poly(1-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride-hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinylchloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon).

In addition, polymers incorporating materials such as chlorosilanes ormethyl-, ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) suchas Dow Chemical Corp. Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical can also be used.

In some methods, elastomers can also be “doped” with uncrosslinkablepolymer chains of the same class. For instance RTV615 can be dilutedwith GE SF96-50 Silicone Fluid. This serves to reduce the viscosity ofthe uncured elastomer and reduces the Young's modulus of the curedelastomer. Essentially, the crosslink-capable polymer chains are spreadfurther apart by the addition of “inert” polymer chains, so this iscalled “dilution”. RTV615 cures at up to 90% dilution, with a dramaticreduction in Young's modulus.

Other examples of doping of elastomer material can include theintroduction of electrically conducting or magnetic species. Should itbe desired, doping with fine particles of material having an index ofrefraction different than the elastomeric material (i.e. silica,diamond, sapphire) is also contemplated as a system for altering therefractive index of the material. Strongly absorbing or opaque particlescan be added to render the elastomer colored or opaque to incidentradiation. This can conceivably be beneficial in an opticallyaddressable system.

Multilayer Construction

Soft lithographic bonding can be used to construct an integrated systemwhich contains multiple channels (e.g., flow channels and/or controlchannels). A heterogenous bonding can be used in which different layersare of different chemistries. For example, the bonding process used tobind respective elastomeric layers together can comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silane (Si—H) groups. The conventional ratiofor RTV 615 is 10A:1B. For bonding, one layer can be made with 30A:1B(i.e. excess vinyl groups) and the other with 3A:1B (i.e. excess silanegroups). Each layer is cured separately. When the two layers are broughtinto contact and heated at elevated temperature, they bond irreversiblyforming a monolithic elastomeric substrate.

A homogenous bonding can also be used in which all layers are of thesame chemistry. For example, elastomeric structures are formed utilizingSylgard 182, 184 or 186, or aliphatic urethane diacrylates such as (butnot limited to) Ebecryl 270 or Irr 245 from UCB Chemical. For example,two-layer elastomeric structures were fabricated from pure acrylatedUrethane Ebe 270. A thin bottom layer was spin coated at 8000 rpm for 15seconds at 170° C. The top and bottom layers were initially cured underultraviolet light for 10 minutes under nitrogen utilizing a Model ELC500 device manufactured by Electrolite corporation. The assembled layerswere then cured for an additional 30 minutes. Reaction was catalyzed bya 0.5% vol/vol mixture of Irgacure 500 manufactured by Ciba-GeigyChemicals. The resulting elastomeric material exhibited moderateelasticity and adhesion to glass.

In some applications, two-layer elastomeric structures were fabricatedfrom a combination of 25% Ebe 270/50% Irr245/25% isopropyl alcohol for athin bottom layer, and pure acrylated Urethane Ebe 270 as a top layer.The thin bottom layer was initially cured for 5 min, and the top layerinitially cured for 10 minutes, under ultraviolet light under nitrogenutilizing a Model ELC 500 device manufactured by Electrolitecorporation. The assembled layers were then cured for an additional 30minutes. Reaction was catalyzed by a 0.5% vol/vol mixture of Irgacure500 manufactured by Ciba-Geigy Chemicals. The resulting elastomericmaterial exhibited moderate elasticity and adhered to glass.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure as described above in FIGS. 18-28, bonding ofsuccessive elastomeric layers can be accomplished by pouring uncuredelastomer over a previously cured elastomeric layer and any sacrificialmaterial patterned thereupon. Bonding between elastomer layers occursdue to interpenetration and reaction of the polymer chains of an uncuredelastomer layer with the polymer chains of a cured elastomer layer.Subsequent curing of the elastomeric layer creates a monolithicelastomeric structure in which a bond is formed between the elastomericlayers.

Referring to the first method of FIGS. 11 to 17B, first elastomericlayer 20 can be created by spin-coating an RTV mixture onmicrofabricated mold 12 at 2000 rpm for 30 seconds yielding a thicknessof approximately 40 microns. Second elastomeric layer 22 can be createdby spin-coating an RTV mixture on microfabricated mold 11. Both layers20 and 22 can be separately baked or cured at about 80° C. for 1.5hours. The second elastomeric layer 22 can be bonded onto firstelastomeric layer 20 at about 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 can be patterned sacrificial layer onsilicon wafers. For example, a Shipley SJR 5740 sacrificial layer can bespun at 2000 rpm, patterned with a high resolution transparency film asa mask and then developed yielding an inverse channel of approximately10 microns in height. When baked at approximately 200° C. for about 30minutes, the sacrificial layer reflows and the inverse channels becomerounded. Optionally, the molds can be treated with trimethylchlorosilane(TMCS) vapor for about a minute before each use in order to preventadhesion of silicone rubber.

Operation of the Microfabricated Components

FIGS. 29A and 29B together show the closing of a flow channel bypressurizing a control channel, with FIG. 29A (a front sectional viewcutting through flow channel 32 in corresponding FIG. 17A), showing anopen flow channel 30; with FIG. 29B showing flow channel 30 closed bypressurization of the control channel 32.

Referring to FIG. 29A, flow channel 30 and control channel 32 are shown.Elastomeric segment 25 separates the channels, forming the top of flowchannel 30 and the bottom of control channel 32. As can be seen, flowchannel 30 is “open”.

As can be seen in FIG. 29B, pressurization of control channel 32 (eitherby gas or liquid introduced therein) causes elastomeric segment 25 todeflect downward, thereby pinching off flow channel 30. Accordingly, byvarying the pressure in control channel 32, an actuable valve system isprovided such that flow channel 30 can be opened or closed by movingelastomeric segment 25 as desired. (For illustration purposes only,channel 30 in FIG. 29B is shown in a “mostly closed” position, ratherthan a “fully closed” position).

It is to be understood that exactly the same valve opening and closingmethods can be achieved with channels 60 and 62. Since such valves areactuated by moving the roof of the channels themselves (i.e., movingelastomeric segment 25), valves and pumps produced by this techniquehave a truly zero dead volume, and switching valves made by thistechnique have a dead volume approximately equal to the active volume ofthe valve, for example, about 100×100×10 μm=100 pL. Such dead volumesand areas consumed by the moving elastomeric segment are approximatelytwo orders of magnitude smaller than known conventional microvalves.Smaller and larger valves and switching valves are contemplated in thepresent invention, and a non-exclusive list of ranges of dead volumeincludes 1 aL to 1 μL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL,and 1 pL to 100 pL

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves of the present invention, volumes ofliquid of 10 nl or smaller can routinely be metered and dispensed. Theaccurate metering of extremely small volumes of fluid enabled by thepresent invention allows chromatography separation of an extremely smallamount of the sample.

FIGS. 30 a and 30 b illustrate valve opening vs. applied pressure for a100 μm wide flow channel 30 and a 50 μm wide control channel 32,respectively. The elastomeric segment of this device was formed by alayer of General Electric Silicones RTV 615 having a thickness ofapproximately 30 μm and a Young's modulus of approximately 750 kPa.FIGS. 30 a and 30 b show the extent of opening of the valve to besubstantially linear over most of the range of applied pressures.

Air pressure was applied to actuate the elastomeric segment of thedevice through a 10 cm long piece of plastic tubing having an outerdiameter of 0.025″ connected to a 25 mm piece of stainless steelhypodermic tubing with an outer diameter of 0.025″ and an inner diameterof 0.013″. This tubing was placed into contact with the control channelby insertion into the elastomeric block in a direction normal to thecontrol channel. Air pressure was applied to the hypodermic tubing froman external LHDA miniature solenoid valve manufactured by Lee Co.

The response of valves of the present invention is substantially linearover a large portion of its range of travel, with minimal hysteresis.While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In some applications, the opening of the valve is usedto control flow rate by being partially actuated to a known degree ofclosure. Linear valve actuation makes it easier to determine the amountof actuation force required to close the valve to a desired degree ofclosure. Another benefit of linear actuation is that the force requiredfor valve actuation can be easily determined from the pressure in theflow channel. If actuation is linear, increased pressure in the flowchannel can be countered by adding the same pressure (force per unitarea) to the actuated portion of the valve.

Control and Pump Systems

FIGS. 31A and 31B show a views of a single on/off valve (e.g., controlsystem), identical to the systems set forth above, (for example in FIG.17A). FIGS. 32A and 32B shows a peristaltic pumping system (e.g., amaterial delivery system) comprised of a plurality of the singleaddressable on/off valves as seen in FIGS. 31A and 31B, but networkedtogether. FIG. 33 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIGS. 32A and32B.

Referring first to FIGS. 31A and 31B, a schematic of channels 30 and 32is shown. Flow channel 30 preferably has a fluid (or gas) flow F passingtherethrough. Control channel 32, which crosses over flow channel 30, ispressurized such that elastomeric segment 25 separating the channels isdepressed into the path of flow channel 30, shutting off the passage offlow F therethrough, as described above.

Referring to FIGS. 32A and 32B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel control channels 32A, 32B and 32C passing thereover. Bypressurizing control line 32A, flow F through flow channel 30 is shutoff under elastomeric segment 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off underelastomeric segment 25B at the intersection of control line 32B and flowchannel 30, etc. Each of control lines 32A, 32B, and 32C is separatelyaddressable. Therefore, peristalsis can be actuated by the pattern ofactuating 32A and 32C together, followed by 32A, followed by 32A and 32Btogether, followed by 32B, followed by 32B and C together, etc. Thiscorresponds to a successive “101, 100, 110, 010, 011, 001” pattern,where “0” indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

Using this process, a pumping rate of 2.35 nL/s was measured bymeasuring the distance traveled by a column of water in thin (0.5 mmi.d.) tubing; with 100×10×10 μm valves under an actuation pressure of 40kPa. As shown in FIG. 24, the pumping rate increased with actuationfrequency until approximately at about 75 Hz, and from about 75 Hz toabove 200 Hz the pumping rate was nearly constant. The valves and pumpsare also quite durable and the elastomeric segment, control channels, orbond have not been observed to fail. Moreover, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations.

Non-Elastomer Based Polymers

As discussed above, while elastomers are preferred materials forfabricating the microfluidic devices of the present invention,non-elastomer based microfluidic devices can also be used in theapparatuses of the present invention. In some applications, thechromatography apparatuses utilize microfluidics based on conventionalmicro-electro-mechanical system (MEMS) technology. Methods of producingconventional MEMS microfluidic systems such as bulk micro-machining andsurface micro-machining have been described, e.g., in Terry et al., AGas Chromatography Air Analyzer Fabricated on a Silicon Wafer, IEEETrans. on Electron Devices, v. ED-26, pp. 1880-1886, 1979; and Berg etal., Micro Total Analysis Systems, New York, Kluwer, 1994, all of whichare incorporated herein by reference in their entirety.

Bulk micro-machining is a subtractive fabrication method whereby singlecrystal silicon is lithographically patterned and then etched to formthree-dimensional structures. For example, bulk micromachiningtechnology, which includes the use of glass wafer processing,silicon-to-glass wafer bonding, has been commonly used to fabricateindividual microfluidic components. This glass-bonding technology hasalso been used to fabricate microfluidic systems.

Surface micro-machining is an additive method where layers ofsemiconductor-type materials such as polysilicon, silicon nitride,silicon dioxide, and various metals are sequentially added and patternedto make three-dimensional structures. Surface micromachining technologycan be used to fabricate individual fluidic components as well asmicrofluidic systems with on-chip electronics. In addition, unlikebonded-type devices, hermetic channels can be built in a relativelysimple manner using channel walls made of polysilicon (see, e.g.,Webster et al., Monolithic Capillary Gel Electrophoresis Stage withOn-Chip Detector, in International Conference on Micro ElectromechanicalSystems, MEMS 96, pp. 491496, 1996), silicon nitride (see, e.g.,Mastrangelo et al., Vacuum-Sealed Silicon Micromachined IncandescentLight Source, in Intl. Electron Devices Meeting, IDEM 89, pp. 503-506,1989), and silicon dioxide.

In some applications, electrokinetic flow based microfluidics can beemployed in the chromatography apparatuses of the present invention.Briefly, these systems direct reagents flow within an interconnectedchannel and/or chamber containing structure through the application ofelectrical fields to the reagents. The electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. Such systems are described, e.g., in WO 96/04547and U.S. Pat. No. 6,107,044.

Electrokinetic flow based microfluidic devices can have a body structurewhich includes at least two intersecting channels or fluid conduits,e.g., interconnected, enclosed chambers, which channels include at leastthree unintersected termini. The intersection of two channels refers toa point at which two or more channels are in fluid communication witheach other, and encompasses “T” intersections, cross intersections,“wagon wheel” intersections of multiple channels, or any other channelgeometry where two or more channels are in such fluid communication. Anunintersected terminus of a channel is a point at which a channelterminates not as a result of that channel's intersection with anotherchannel, e.g., “T” intersection.

In some electrokinetic flow based apparatuses, at least threeintersecting channels having at least four unintersected termini arepresent. In a basic cross channel structure, where a single horizontalchannel is intersected and crossed by a single vertical channel,controlled electrokinetic transport operates to direct reagent flowthrough the intersection, by providing constraining flows from the otherchannels at the intersection. Simple electrokinetic flow of this reagentacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage).

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1-14. (Canceled)
 15. A method for producing a microfluidicchromatography apparatus comprising: (a) producing a microfabricatedfluid delivery system from a material comprising an elastomeric polymer,wherein the fluid deliver system comprises: (i) a microfluidic flowchannel comprising a flow channel inlet for introducing the fluid intosaid flow channel and a flow channel outlet, (ii) a flow controlchannel, (iii) a flow control valve comprised of a flow controlelastomeric segment that is disposed in between said flow channel andsaid flow control channel to regulate fluid flow through said flowchannel, wherein said flow control valve is deflectable into orretractable from said flow channel upon which said flow control valveoperates in response to an actuation force applied to said flow controlchannel, said flow control elastomeric segment when positioned in saidflow channel restricting fluid flow therethrough, and (iv) a flowcontrol channel actuation system operatively interconnected to said flowcontrol channel for applying an actuation force to said flow controlchannel; and (b) connecting the fluid delivery system to achromatography column having a column inlet and a column outlet suchthat the column inlet is in fluid communication with the flow channeloutlet, wherein the chromatography column comprises a stationary phasewhich is capable of separating at least a portion of the analyte in thefluid.
 16. The method of claim 15, wherein the flow channel is locatedon an interface between a solid substrate and the elastomeric polymersuch that an inner surface of the flow channel comprises an elastomericpolymer portion and a solid substrate portion.
 17. The method of claim16, wherein the stationary phase is attached to the solid substrateportion of the flow channel inner surface.
 18. The method of claim 17,wherein the elastomeric polymer portion of the flow channel innersurface comprises a surface coating that reduces a non-specific bindingof the analyte.
 19. The method of claim 15 further comprising: (a)microfabricating the chromatography column which comprises achromatography channel having an inner surface which comprises afunctional group; and (b) attaching a stationary phase compound to atleast a portion of the inner surface by reacting the stationary phasecompound with the functional group under conditions sufficient to form acovalent bond between the functional group and the stationary phasecompound.
 20. The method of claim 19, wherein the functional group issilane.
 21. The method of claim 20, wherein the stationary phasecompound is 1-octadecene.
 22. The method of claim 19, wherein themicrofabricated chromatography column further comprises amicrofabricated rotary channel comprising: a rotary channel inlet; arotary channel outlet; a rotary control channel; a rotary inlet controlvalve comprised of an elastomeric segment of said rotary inlet controlchannel that is disposed in between said rotary channel inlet and saidrotary control channel to regulate fluid flow into said rotary channel,wherein said rotary inlet control valve is deflectable into orretractable from said rotary channel inlet upon which said rotary inletcontrol valve operates in response to an actuation force applied to saidrotary control channel, said elastomeric segment of said rotary inletcontrol channel when positioned in said rotary channel inlet restrictingfluid flow therethrough; a rotary outlet control valve comprised of anelastomeric segment of said rotary outlet control channel that isdisposed in between said rotary channel outlet and said rotary controlchannel to regulate fluid flow out of said rotary channel, wherein saidrotary outlet control valve is deflectable into or retractable from saidrotary channel outlet upon which said rotary outlet control valveoperates in response to an actuation force applied to said rotarycontrol channel, said elastomeric segment of said rotary control channeloutlet when positioned in said rotary channel outlet restricting fluidflow therethrough; a rotary pump valve comprised of an elastomericsegment of said rotary pump that is disposed in between said rotarychannel and said rotary pump control channel to regulate fluid flowthrough said rotary channel, wherein said rotary pump valve isdeflectable into or retractable from said rotary channel upon which saidrotary pump valve operates in response to an actuation force applied tosaid rotary pump control channel, said elastomeric segment of saidrotary pump when positioned in said rotary channel restricting fluidflow therethrough; and a rotary control channel actuation systemoperatively interconnected to said rotary control channel for applyingan actuation force to said rotary control channel.
 23. A method forseparating an analyte from a sample fluid comprising: (A) introducingthe sample fluid into a microfluidic chromatography apparatuscomprising: (a) a microfabricated fluid delivery system which isproduced from a material comprising an elastomeric polymer, wherein saidfluid deliver system comprises: (i) a microfluidic flow channelcomprising a flow channel inlet for introducing the fluid into said flowchannel and a flow channel outlet, (ii) a flow control channel, (iii) aflow control valve comprised of a flow control elastomeric segment thatis disposed in between said flow channel and said flow control channelto regulate fluid flow through said flow channel, wherein said flowcontrol valve is deflectable into or retractable from said flow channelupon which said flow control valve operates in response to an actuationforce applied to said flow control channel, said flow controlelastomeric segment when positioned in said flow channel restrictingfluid flow therethrough, and (iv) a flow control channel actuationsystem operatively interconnected to said flow control channel forapplying an actuation force to said flow control channel; and (b) achromatography column comprising (i) a stationary phase which is capableof separating at least a portion of the analyte from the sample fluid,(ii) a column inlet which is in fluid communication with the flowchannel outlet, and (iii) a column outlet through which a separatedfluid exits the chromatography column; and (B) eluting the sample fluidthrough the chromatography column with an eluent to separate at least aportion of the analyte.
 24. The method of claim 23, wherein fluid flowthrough the chromatography column is achieved by a peristaltic pumpaction created by actuating one or more of the flow control valves. 25.The method of claim 24, wherein the chromatography column comprises amicrofabricated rotary channel comprising: a rotary channel inlet; arotary channel outlet; a rotary control channel; a rotary inlet controlvalve comprised of an elastomeric segment of said rotary inlet controlchannel that is disposed in between said rotary channel inlet and saidrotary control channel to regulate fluid flow into said rotary channel,wherein said rotary inlet control valve is deflectable into orretractable from said rotary channel inlet upon which said rotary inletcontrol valve operates in response to an actuation force applied to saidrotary control channel, said elastomeric segment of said rotary inletcontrol channel when positioned in said rotary channel inlet restrictingfluid flow therethrough; a rotary outlet control valve comprised of anelastomeric segment of said rotary outlet control channel that isdisposed in between said rotary channel outlet and said rotary controlchannel to regulate fluid flow out of said rotary channel, wherein saidrotary outlet control valve is deflectable into or retractable from saidrotary channel outlet upon which said rotary outlet control valveoperates in response to an actuation force applied to said rotarycontrol channel, said elastomeric segment of said rotary control channeloutlet when positioned in said rotary channel outlet restricting fluidflow therethrough; a rotary pump valve comprised of an elastomericsegment of said rotary pump that is disposed in between said rotarychannel and said rotary pump control channel to regulate fluid flowthrough said rotary channel, wherein said rotary pump valve isdeflectable into or retractable from said rotary channel upon which saidrotary pump valve operates in response to an actuation force applied tosaid rotary pump control channel, said elastomeric segment of saidrotary pump when positioned in said rotary channel restricting fluidflow therethrough; and a rotary control channel actuation systemoperatively interconnected to said rotary control channel for applyingan actuation force to said rotary control channel.
 26. The method ofclaim 25 further comprising: introducing at least a portion of thesample fluid into the rotary channel; closing the rotary inlet and therotary outlet control valves by actuating the rotary inlet and therotary outlet control valves; transporting the sample fluid through therotary channel by actuating one or more of the rotary pump valves untilat least a portion of the analyte is adsorbed onto the stationary phase;opening the rotary inlet and rotary outlet control channels; introducinga first eluent through the rotary inlet channel and removing theresulting mixture through the rotary outlet channel, wherebysubstantially all of the sample fluid is removed from the rotary channeland at least about 95% of the adsorbed analyte remains adsorbed onto thestationary phase; and introducing a second eluent, which is capable ofremoving the analyte from the stationary phase, through the rotary inletchannel and removing the resulting mixture through the rotary outletchannel, whereby substantially all of the adsorbed analyte is removedfrom the rotary channel.
 27. The method of claim 26, wherein saidanalyte is a protein having a molecular weight of at least about 1000g/mol.
 28. The method of claim 27, wherein the stationary phase is C-18alkyl and the sample fluid is an aqueous solution.
 29. The method ofclaim 28, wherein the first eluent is selected from the group consistingof water and an aqueous buffer solution.
 30. The method of claim 29,wherein the second eluent comprises an organic solvent selected from thegroup consisting of an alcohol, acetonitrile, dimethylformamide, andmixtures thereof.
 31. The method of claim 30, wherein the second eluentis a mixture of the organic solvent and water or an aqueous buffersolution.